Tissue engineering is a rapidly developing field of biomedical research that aims at repair, replace or regenerate damaged tissues. Due to the latest events of disastrous phase II clinical drug trials (e.g. TGN1412), further goals of tissue engineering include generation of human tissue models for safety and efficacy testing of pharmaceutical compounds. Tissue engineering in general and our model in particular exploits biological morphogenesis, which is an example of self-assembly.
Cell and tissue engineering is pursued nowadays from several perspectives. In one aspect, it is used to gain deeper insight into cell biology and physiology.
Three Dimensional Tissue Models
Two main directions are being developed in tissue engineering research: tissue scaffold based and tissue scaffold free systems. While scaffold based systems use mostly biodegradable scaffold material to provide an artificial 3D structure to facilitate cellular interactions, a scaffold free system allows direct cell to cell interactions, and allows cells to grow on their secreted scaffold material in the model.
In US 2001/0055804 A1 (“Three dimensional in vitro model of human preneoplastic breast disease”) discloses a three dimensional in vitro cell culture system useful as a model of a preneoplastic breast disease for screening drugs. Said model is prepared by co-culturing preneoplastic epithelial cells of breast origin, endothelial cells and breast fibroblasts on a reconstituted base membrane in a medium comprising further additives like growth factors, estrogens etc. Thus, in this solution a base membrane, preferably Matrigel® is used as a tissue scaffold. According to the description a network of branching ductal alveolar units vasculature is formed within about 3-7 days in this system.
The system relates to breast and not lung and there is no suggestion to make a lung culture by that method. In US 2003/0109920 (“Engineered animal tissue”) microvascular endothelial cells were obtained from adult lung and placed between two layers of human dermal fibroblasts present in a three dimensional collagen gel. Thus, a sandwich structure was formed.
Though the vascular endothelial cells were obtained from human lung, the artificial tissue prepared by this method was similar to human skin, therefore, it can not be actually considered as a pulmonary tissue model. In US 2008/0112890 (“Fetal pulmonary cells and uses thereof”) a 3D tissue-like preparation is taught which is based on fetal mouse epithelial, endothelial and mesenchymal cells. The authors used mouse embryonic lung cells to the preparation in order to obtain lung prosthesis and perform screening on the 3D tissue-like preparation. As a matrix, a hydrogel, MATRIGEL™ was used to establish appropriate cell-cell interactions. It appears from the description that a fibroblast overgrowth was experienced upon coculturing epithelial cells and fibroblasts.
WO2008/100555 (“Engineered lung tissue construction for high throughput toxicity screening and drug discovery”) relates to a lung tissue model preparation comprising fetal pulmonary cells and a tissue scaffold made of a biocompatible material and preferably a fibroblast growth factor. Fetal pulmonary cells comprise epithelial, endothelial and mesenchymal cells. A number of applicable biocompatible materials are listed.
WO2004/046322 (“Replication of biological tissue”) preparation of an artificial 3-D tissue is proposed under microgravity environment. The tissue is based on human breast cancer cells and is useful as a breast cancer model. In a rotating bioreactor chamber at first connective tissue cells are cultured till the formation of a 3-D spheroid structure, then sequentially endothelial and epithelial cells are added to the culture. It is to be noted that the teaching is theoretical and while protocols are provided to culture the cells and to handle to cultures, no actual results of the experiments are disclosed.
Vertrees, R A, Zwischenberger, J B et al. (2008) cultured mesenchymal cells (human bronchial tracheal cells) on collagen coated microporous cyclodextrin beads (Cytodex-3 micro carrier beads) in rotating walled vessels. After 24 to 48 h a malignant phenotype human epithelial cell line (BZR-T33) was added. The purpose of the authors was to develop a 3-D model of lung cancer. The authors observed that in the prior art primary cell lines “of human broncho-epithelial cells and human lung cancers provide a more differentiated model similar to the structure and function of epithelial cells in vivo; however, these models are short-lived in vitro.”
Nichols, J. E. and Cortiella, J (2008) review recent advancements toward the development of a tissue-engineered lung and find that hitherto developed lung tissue models used specialized scaffold materials and were kept in culture lengthily. The authors mention that “[P]roblems to be faced in the development of . . . lung, depend on . . . the development of appropriate scaffolds and matrices to enhance and support three-dimensional (3D) production of tissues.
Molnar T. F. and Pongracz J. E. provide an overview of different applications of tissue engineering among others in lung tissue cultures. The authors note that “3D cultures . . . create additional problems, including difficulties of gas and nutrient exchange in the third dimension. To circumvent this problem, scaffolds . . . are purposefully engineered in a way to make tissue build-up and allow cellular interactions, nutrients and gas exchange to occur.” They conclude, however, that “Biotechnology in reconstructive surgery is still a test-tube based laboratory issue . . . ”
Scaffoldless designs are not envisioned in these two reviews.
Wnt Signaling and Wnt11
Wnt proteins are secreted morphogens that are required for basic developmental processes as well as maintenance of adult tissue homeostasis. In the lung, Wnt signaling controls proliferation, cell-fate decision, maturation and terminal differentiation of progenitor cells. Alveolar type II (ATII) cells are progenitors of alveolar type I (ATI) cells that create the primary gas exchange surface of the alveoli. However, how ATII type differentiation is regulated is still far from understood.
Wnt signaling regulates a variety of developmental processes [Dobbs, L. G. (1989)]. Over-expression (Wnt13 and Wnt2,4) or down-regulation (Wnt7a,5) of Wnts or Wnt pathway inhibitors (Dkk3,6, WIF7 and sFRP8), are characteristic in different types of pulmonary diseases including fibrosis and tumors. Additionally, over-expression of disheveled (Dvl), a signal transducer of Wnt signals, has been reported in 75% of NSCLC cases, highlighting the importance of Wnt pathways in pulmonary cancers.
At least three signaling pathways are involved in the signal transduction process: the canonical or β-catenin dependent, and two non-canonical pathways. Epithelial-mesenchymal transitition (EMT) is generally linked to increased β-catenin dependent signaling activity in many invasive or metastatic tumors [Voulgari, A. and Pintzas, A (2009); Zeisberg, M. and Neilson, E. G. (2009)]. Although β-catenin mutations in lung cancers are relatively rare, upregulation of uncomplexed β-catenin without genetic alteration to β-catenin itself has been shown in a high proportion of human NSCLCs [Akiri G. et al. (2009)]. β-catenin dependent signaling is also essential in pulmonary regeneration. Thus, a balance in Wnt levels seems to be essential to keep the healthy equilibrium in pulmonary homeostasis.
The shortcomings of non-human models in mimicking human tissue characteristics often compromise the success of understanding molecular interactions in human tissues. Recently, therefore primary human cells are frequently used in molecular studies. In traditional two dimensional cell cultures, however, primary cells loose their characteristic differentiation markers [Shannon, J. M., (1987)]. The present inventor have not information about any art study on the effect of Wnt11 in 3D model tissue cultures.
Despite the extensive literature of lung models, it appears that the prior art discloses only tissue scaffold based three dimensional models and no simple, tissue scaffold free lung tissue model, comprising at least epithelial cells and fibroblasts, is disclosed in the prior art.
The present Inventors have surprisingly found that by simple biochemical methods a tissue scaffold free lung tissue model system can be created which has certain morphological features of lung tissue and is in several aspects more favorable than two dimensional (2D) systems or systems based on a matrix. The model of the invention avoids the problem of fibroblast overgrowth. The present inventors also recognized and provide the first evidence that Wnt11 is a regulator of ATII type differentiation. Thus, due to the effect of Wnt11 the model can be further improved both in terms of marker expression and morphology. Wnt11 is also shown to be down-regulated in lung cancers (LCs). Thus, according to the invention a pulmonary cancer model is provided wherein expression of Wnt11 is down-regulated in the mesenchymal cells of the model tissue culture.
Moreover, the present Inventors recognized that Wnt11 is useful in repair and/or regeneration of tissue, preferably lung tissue after injury.
The pulmonary model tissue culture can be prepared rapidly. The present invention provides a model tissue which is ready for use in various tests. The model is suitable to study cell-cell interactions in various lung tissues to mimic normal function and disease development.